Normal induction but attenuated progression of germinal center responses in BAFF and BAFF-R signaling-deficient mice - PubMed (original) (raw)

Normal induction but attenuated progression of germinal center responses in BAFF and BAFF-R signaling-deficient mice

Ziaur S M Rahman et al. J Exp Med. 2003.

Abstract

The factors regulating germinal center (GC) B cell fate are poorly understood. Recent studies have defined a crucial role for the B cell-activating factor belonging to TNF family (BAFF; also called BLyS) in promoting primary B cell survival and development. A role for this cytokine in antigen-driven B cell responses has been suggested but current data in this regard are limited. A BAFF receptor expressed by B cells (BAFF-R/BR3) is defective in A/WySnJ mice which exhibit a phenotype similar to BAFF-deficient (BAFF-/-) animals. Here, we show that although GC responses can be efficiently induced in both A/WySnJ and BAFF-/- mice, these responses are not sustained. In BAFF-/- mice, this response is rapidly attenuated and accompanied by perturbed follicular dendritic cell development and immune complex trapping. In contrast, analysis of the A/WySnJ GC response revealed a B cell autonomous proliferative defect associated with reduced or undetectable Ki67 nuclear proliferation antigen expression by GC B cells at all stages of the response. These data demonstrate a multifaceted role for the BAFF pathway in regulating GC progression.

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Figures

Figure 1.

Figure 1.

B cell areas and phenotype in the spleens of BAFF−/− and A/WySnJ mice. Spleens of unimmunized BAFF−/−, C57BL/6, A/WySnJ, and A/J mice were processed for histology and flow cytometry and stained with the indicated reagents. Representative splenic areas in adjacent sections (A) and flow cytometric plots (B) are illustrated for each strain. Due to strain differences in average levels of sIgD revealed in the A/J and C57BL/6 samples, the gates used to define the sIgMlow sIgDhigh subpopulation in A/WySnJ and BAFF−/− were set at different positions. Flow cytometry data were obtained from the pooled spleens of two mice of each genotype.

Figure 1.

Figure 1.

B cell areas and phenotype in the spleens of BAFF−/− and A/WySnJ mice. Spleens of unimmunized BAFF−/−, C57BL/6, A/WySnJ, and A/J mice were processed for histology and flow cytometry and stained with the indicated reagents. Representative splenic areas in adjacent sections (A) and flow cytometric plots (B) are illustrated for each strain. Due to strain differences in average levels of sIgD revealed in the A/J and C57BL/6 samples, the gates used to define the sIgMlow sIgDhigh subpopulation in A/WySnJ and BAFF−/− were set at different positions. Flow cytometry data were obtained from the pooled spleens of two mice of each genotype.

Figure 2.

Figure 2.

Kinetics of germinal center (GC) induction. (A) A/J (▪) and A/WySnJ (□) mice and (B) BAFF−/− (○) and C57BL/6 mice (•, BAFF+/+) were immunized with SRBC. Two spleen sections per mouse obtained at each time point, each at least 40–50 sections apart, were subjected to immunohistological analysis. Small, medium, and large PNA+ GCs were counted per 10× field. The data represent at least three mice from each group at each time point.

Figure 3.

Figure 3.

Immunofluorescent staining of B cells, FDCs, and T cells in GCs. Data from day 12 of the SRBC response in A/J and A/WySnJ mice and those from day 6 in BAFF−/− and C57BL/6 are shown. Staining was performed (A) with mAbs against GL7 (green) and FDC-M1 and 2.4G2 (both in red), (B) with GL7 (green) and CD35 (CR1) in red, (C) with anti–TCR-C β (red) and GL7 (green), and (D) with polyclonal antiserum against Bcl-6 (red). The data are representative of three mice from each group. Original magnification of images was 25×.

Figure 4.

Figure 4.

Immune complex trapping in A/WySnJ and BAFF−/− GCs. A/WySnJ, A/J, BAFF−/−, and C57BL/6 mice were immunized with SRBC and subsequently injected with HRP and anti-HRP to evaluate IC trapping as described in Materials and Methods. The figure illustrates representative splenic areas in adjacent sections stained either with anti-CD35 to reveal the follicular reticulum or with an anti-HRP antibody. Original magnification of images was 10×. Arrows indicate areas of IC deposition on the follicular reticulum.

Figure 5.

Figure 5.

Quantitation of apoptotic nuclei in GCs at multiple time points. Eight randomly chosen small and medium GCs from two to three A/J (•) and A/WySnJ (○) mice at each time point of the SRBC response were assessed for apoptotic activity by enumerating TUNEL-positive nuclei. Horizontal bars represent mean values.

Figure 6.

Figure 6.

(A) In situ BrdU incorporation assay in A/J and A/WySnJ GCs. One of two parallel sections obtained from SRBC-immunized mice was stained (top panel, for day 9 and 14) with anti-IgD (blue) and PNA (red), and the other section (bottom panel, for day 9 and day 14) was stained with anti-BrdU (red). Data are representative of three mice at each time point. (B) PI cell cycle analysis on GC B cells (B220+GL7+IgD−) obtained by FACS® on day 8 post-SRBC immunization. The horizontal bar and number in each panel represent GC B cells that are in S/G2-M phases. These data were obtained from the pooled spleen cells of three A/J and five A/WySnJ mice. Original magnification of images was 25×.

Figure 6.

Figure 6.

(A) In situ BrdU incorporation assay in A/J and A/WySnJ GCs. One of two parallel sections obtained from SRBC-immunized mice was stained (top panel, for day 9 and 14) with anti-IgD (blue) and PNA (red), and the other section (bottom panel, for day 9 and day 14) was stained with anti-BrdU (red). Data are representative of three mice at each time point. (B) PI cell cycle analysis on GC B cells (B220+GL7+IgD−) obtained by FACS® on day 8 post-SRBC immunization. The horizontal bar and number in each panel represent GC B cells that are in S/G2-M phases. These data were obtained from the pooled spleen cells of three A/J and five A/WySnJ mice. Original magnification of images was 25×.

Figure 7.

Figure 7.

Staining of GC B cells for Ki67, a nuclear proliferation marker. Spleen sections from days 6, 9, and 12 post-SRBC immunization were stained with anti-Ki67 (red) and GL7 (green). (A) GCs were categorized into bright (+/+), moderately positive (+/−), and substantially reduced or undetectable (−/−) levels of Ki67 staining. (B) Ki67 expression in A/WySnJ and A/J control and BAFF−/− and C57BL/6 GCs at different time points (C) Percentage of A/J and A/WySnJ GCs in each category. These data represent 35–50 randomly selected GCs from five mice from each group at each time point. Original magnification of images was 25×. (D) The percentage of Ki67+ cells in resting (0 h) and in in vitro anti-CD40 and IL-4–stimulated (48 h) splenic B cells purified from A/J (hatched bar) and A/WySnJ (white bar) mice.

Figure 7.

Figure 7.

Staining of GC B cells for Ki67, a nuclear proliferation marker. Spleen sections from days 6, 9, and 12 post-SRBC immunization were stained with anti-Ki67 (red) and GL7 (green). (A) GCs were categorized into bright (+/+), moderately positive (+/−), and substantially reduced or undetectable (−/−) levels of Ki67 staining. (B) Ki67 expression in A/WySnJ and A/J control and BAFF−/− and C57BL/6 GCs at different time points (C) Percentage of A/J and A/WySnJ GCs in each category. These data represent 35–50 randomly selected GCs from five mice from each group at each time point. Original magnification of images was 25×. (D) The percentage of Ki67+ cells in resting (0 h) and in in vitro anti-CD40 and IL-4–stimulated (48 h) splenic B cells purified from A/J (hatched bar) and A/WySnJ (white bar) mice.

Figure 7.

Figure 7.

Staining of GC B cells for Ki67, a nuclear proliferation marker. Spleen sections from days 6, 9, and 12 post-SRBC immunization were stained with anti-Ki67 (red) and GL7 (green). (A) GCs were categorized into bright (+/+), moderately positive (+/−), and substantially reduced or undetectable (−/−) levels of Ki67 staining. (B) Ki67 expression in A/WySnJ and A/J control and BAFF−/− and C57BL/6 GCs at different time points (C) Percentage of A/J and A/WySnJ GCs in each category. These data represent 35–50 randomly selected GCs from five mice from each group at each time point. Original magnification of images was 25×. (D) The percentage of Ki67+ cells in resting (0 h) and in in vitro anti-CD40 and IL-4–stimulated (48 h) splenic B cells purified from A/J (hatched bar) and A/WySnJ (white bar) mice.

Figure 7.

Figure 7.

Staining of GC B cells for Ki67, a nuclear proliferation marker. Spleen sections from days 6, 9, and 12 post-SRBC immunization were stained with anti-Ki67 (red) and GL7 (green). (A) GCs were categorized into bright (+/+), moderately positive (+/−), and substantially reduced or undetectable (−/−) levels of Ki67 staining. (B) Ki67 expression in A/WySnJ and A/J control and BAFF−/− and C57BL/6 GCs at different time points (C) Percentage of A/J and A/WySnJ GCs in each category. These data represent 35–50 randomly selected GCs from five mice from each group at each time point. Original magnification of images was 25×. (D) The percentage of Ki67+ cells in resting (0 h) and in in vitro anti-CD40 and IL-4–stimulated (48 h) splenic B cells purified from A/J (hatched bar) and A/WySnJ (white bar) mice.

Figure 8.

Figure 8.

Reduced Ki67 expression in A/WySnJ GCs is due to a B cell autonomous defect. Spleen sections of A/J + μMT → AB6F1 and A/WySnJ + μMT → AB6F1 chimeric mice from day 9 post-SRBC immunization were stained with (A) anti-B220 (blue) and GK1.5-HRP (red), (B) GL7-FITC (green) and FDC-M1 (red), (C) GL7-FITC (green) and Ki 67 (red), and (D) GL7-FITC (green) and TCRC-β-PE (red). Original magnification of images was 10× in A and 25× in B–D. The data represent one A/J + μMT → AB6F1 and two A/WySnJ + μMT → AB6F1 chimeras.

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